THE RELATION BETWEEN THE AXON INITIAL SEGMENT WITH SPONTANEOUS AND DRIVEN FIRING OF SUBSTANTIA NIGRA DOPAMINERGIC NEURONS

Abstract

The axonal region most proximal to soma is the axon initial segment. This region is enriched in voltage-gated sodium channels as well as scaffolding proteins such as ankyrin-G, which allow to this structure to initiate action potentials. As shown in other central neurons, the molecular composition, size and position of the axon initial segment is known to influence the neuron’s activity. In order to describe the mechanisms that influence the characteristic pacemaker firing of individual substantia nigra pars compacta dopaminergic neurons, we firstly identified and structurally characterized the axon initial segment. Then, we examined whether the structural characteristics of the axon initial segment relate to the spontaneous and driven firing showed by these neurons in vivo. To test this hypothesis, dopaminergic neurons were juxtacellularly labeled in vivo, the axon initial segments were identified using immunoreactivity against ankyrin-G and then they were located in the 3D reconstruction of the labeled neurons. The results showed that the axon initial segments of dopaminergic neurons varied in size and geometry. The diameter of the axon initial segment decreases towards its distal border, suggesting a distal locus of generation of action potentials in these neurons, probably triggered by sodium channels containing the subunit 1.2. On the other hand, spontaneous firing rate of neurons increased with the size of axon initial segments and decreased with soma to axon initial segment distance. Thus, neurons with more proximal and larger axon initial segments fire faster than neurons with more distal and smaller axon initial segments. Notably, the spontaneous firing rate and both the size as well as the position of the axon initial segment do not correlate with the somatodendritic size or the size of the soma and dendrites separately. In order to determine how the structure of axon initial segment could influence spontaneous firing rate, and as part of an international collaboration, a model of spontaneously firing dopaminergic neuron based in pacemaking mechanism was implemented by using our 3D reconstructed dopaminergic neurons. The data indicate that the spontaneous firing rate is reached by a frequency consensus between the activity of the axon initial segment and the somatodendritic compartment: the larger the axon initial segment in relation to the somatodendritic compartment, the higher spontaneous firing rate. Finally, the aversive-driven activity, but not the spontaneous firing rate, was correlated with the inhibitory synaptic innervation onto the proximal axon-bearing dendrites, which are close to the axon initial segment. Neurons that were inhibited by the aversive stimulus had a higher density of inhibitory synapses onto proximal axon-bearing dendrite than neurons that did not respond. The results exposed in the present thesis represent an important progress in the understanding of the morphological and synaptic basis that underlie the spontaneous and driven activity of dopaminergic neurons, which in turn are crucial to maintaining baseline dopamine levels and neuronal responses to motivationally relevant stimuli